Kay Powell with Richard Vierstra PhD, Xiaohua Zheng and Joe Walker PhD

UW-Madison Department of Genetics

Plant DNA was screened for homozygous T-DNA insertions in F-box genes in order to generate a set of plant insertion lines having reduced or null expression of individual genes with the end goal of gaining a better understanding of F-box gene function. Agrobacterium tumefaciens was used to create the T-DNA insertion library in Arabidopsis Thaliana. F-box proteins help identify target proteins for degradation by binding to their respective target proteins and attaching the marker protein ubiquitin. The function of a particular F-box gene can be inhibited in any individual plant that is homozygous with respect to a T-DNA insertion within that gene.

Arabidopsis seeds containing T-DNA insertions were planted and grown for 10-15 days.  DNA was extracted from the leaves and individual genes and their corresponding T-DNA insertion sequences were amplified using the polymerase chain reaction (PCR) method with primer combinations specific to each gene and the T-DNA insertion sequence.  A total of 73 lines were screened. Forty-two of these lines had individuals that were homozygous for their mutations. These plants were transferred to soil for full growth and phenotypic observation.